Although the interferon-γ release assay (IGRA) has become a widely accepted means for the diagnosis of latent tuberculosis infection (LTBI), the role of the IGRA in diagnosing active tuberculosis (ATB) among human immunodeficiency virus (HIV)-seropositive individuals remains controversial. Previous analyses did not set up rational inclusive criteria for screening articles with strict control groups and a gold standard for ATB diagnosis. Therefore, we conducted a systematic review of the latest evidence to evaluate the accuracy of IGRA for HIV-seropositive patients.Initially, we searched the EMBASE, Cochrane and MEDLINE databases to find research articles published from January 2000 to October 2015 that used the QuantiFERON-TB Gold In-Tube assay (QFT-IT) or the T-SPOT.TB assay (T-SPOT) to diagnose ATB among HIV-seropositive individuals. We separately calculated the pooled sensitivity, specificity, and proportion of indeterminate events and then summarized the results using forest plots to estimate the accuracy of the QFT-IT and T-SPOT assays.2 = 85.22 %) and 76 % , respectively, and the optimum area under the curve (AUC) was 0.78 . The pooled sensitivity and specificity of the T-SPOT assay were 89 %  and 87 % , respectively, and the AUC was 0.93 . The pooled ratios of the indeterminate results of the QFT-IT and T-SPOT assays were 0.07  and 0.19 , respectively, calculated using the fixed effect model, and 0.08  and 0.10 , respectively, calculated using the random effects model.A total of 1,743 studies were discovered after searching; 11 studies met our selection standards and were included for meta-analysis. The pooled sensitivity and specificity of the QFT-IT assay were 69 % (95 % CI, 50–84 %, IThe IGRA does not appear to be optimal for the clinical confirmation of ATB cases in HIV-seropositive patients; however, the T-SPOT assay may have greater accuracy in distinguishing ATB cases among HIV-infected individuals than the QFT-IT assay, while the QFT-IT assay appears to reduce the occurrence of indeterminate results. Furthermore, modification and additional trial designs are required to improve diagnostic effectiveness. Mycobacterium tuberculosis infection has become one of the leading causes of mortality among patients living with acquired immunodeficiency syndrome (AIDS) caused by infection with the human immunodeficiency virus (HIV). In fact, one third of deaths among HIV-seropositive patients are due to tuberculosis (TB). The World Health Organization (WHO) reported 9.6 million TB cases worldwide in 2014 and 1.2 million HIV-seropositive TB cases. These cases were especially prominent in Africa, where HIV-seropositive TB cases represented 74 % of the total HIV/TB co-infections  and an enzyme-linked immunosorbant assay   . HoweverWe conducted a comprehensive systematic review and meta-analysis of the latest evidence to evaluate the accuracy of IGRA in diagnosing ATB among HIV-infected patients to guide the application of the IGRA in immunocompromised population.Mycobacterium tuberculosis culture was adopted as the gold standard for the diagnosis of ATB in this systematic review.This systematic review and meta-analysis was conducted within the guidelines of the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement . BecauseWe systematically searched all studies that estimated the accuracy of the IGRA  for ATB among HIV-seropositive adult individuals. We searched the EMBASE, Cochrane and MEDLINE databases to find research articles published from January 1, 2000 to October 30, 2015. Searches were implemented using combinations of the following terms: “tuberculosis”, “active tuberculosis”, “interferon-gamma release assay”, “QFT-IT”, “T-SPOT”, “CFP-10”, “ESAT-6”, “IGRA”, “acquired immunodeficiency syndrome”, “human immunodeficiency virus” and “HIV”. We searched for additional references from review articles, guidelines and conferences when necessary.Mycobacterium tuberculosis culture; 4) the number of HIV-seropositive individuals was less than 10; 5) studies where screened individuals had received anti-TB therapy; 6) repeat research by the same author; 7) original data was not included; and 8) reports of prospective studies, conference abstracts, reviews, guidelines, letter and case reports. All citations were screened and verified independently by two investigators. Group discussion took place when the two investigators disagreed on the inclusive criteria of a citation.In this systematic review, two investigators (ZY. Huo and L. Peng) independently completed a primary scan to screen references that were potentially appropriate based on the titles and abstracts. Studies that only evaluated the performance of IGRA for diagnosing active TB in HIV-seropositive patients on blood samples were included. Then, a secondary selection was completed by reviewing the full text of the article. Articles were excluded if one of the follow criteria was fulfilled: 1) the study did not set up a non-ATB control group in HIV-seropositive patients; 2) experiments were performed with an older generation of the IGRA method, did not use a commercial IGRA, or the cut-off values of the IGRA were not the same; 3) ATB cases were not confirmed by Data from all included research studies were extracted and crosschecked by two investigators independently. The following data from English-language articles were extracted: 1) author, publication year of article, calendar period of research, and the nation where the research was performed; 2) the age group of the enrolled participants; 3) the type of commercial IGRA studied; 4) the proportion of ATB among HIV-seropositive participants diagnosed by culture; and 5) the amount of indeterminate, negative and positive results for each group among the participants. The quality of all included studies was evaluated using the quality assessment of diagnostic accuracy studies-2 (QUADAS-2) checklist, which is a validated and widely used criterion for the diagnostic accuracy of trials .Mycobacterium tuberculosis diagnosis by direct culture were classified as ATB patients, and the remaining cases were assumed to be non-ATB patients. Indeterminate results were excluded prior to calculating sensitivity and specificity if reported [2 statistic to examine the influence of authentic variability instead of errors associated with diagnostic sampling. The separated sensitivity and specificity of a single test for each study were used to generate a symmetric receiver operator characteristic (SROC) curve, which summarized the overall test performance and represented the variation occurring in the diagnostic accuracy among studies [We adopted the standard methods recommended for systematic meta-analyses of diagnostic test evaluations , 13. Stareported –16. Stat studies –19.3, except for three studies that did not report the median CD4+ counts in the text. Additional specific information with respect to the studies included in this systematic review is available upon request.A total of 1,743 studies were identified after searching in the EMBASE, Cochrane and MEDLINE databases. After the full text of the articles was assessed for eligibility, 11 studies met our selection standards and were included in the meta-analysis –30. FiguFigure 2 = 85.22 %), and the pooled specificity was 76 % , whereas the pooled specificity of the T-SPOT assay was 87 %  and the small number of inclusive studies.At the same time, the combined sensitivity of the T-SPOT assay was calculated from four studies with a total of 444 individuals after excluding the indeterminate events –30. The 2 = 74.8 %) when using the fixed effect model; when using the random effects model, the pooled ratio of the QFT-IT assay was 0.08 ; the pooled ratio for the T-SPOT assay was 0.10  when using the random effects model . The pooled ratio of QFT-IT indeterminate events was 0.07 , which suggested a low risk of publication bias for this assay. However, Fig. P = 0.04), which indicates a potential risk of publication bias.The publication bias of the 11 included studies was evaluated and analyzed using Deeks’ funnel plot asymmetry test Figs.  and 10. The results of our systematic review, which differ from previous analyses, appear to suggest that the IGRA plays a limited role in diagnosing ATB in HIV-seropositive patients. However, this meta-analysis is an update and improves on previous articles in several respects , 32. ComAs shown in Fig. The pooled sensitivity and specificity of the QFT-IT assay were 69 and 76 %, respectively, for diagnosing ATB among HIV-seropositive patients after excluding indeterminate results, which were similar to the results of previous studies in which the sensitivity and specificity varied from 61–76.7 to 72–76.1 %, respectively , 32. TheThe pooled ratios of the indeterminate QFT-IT and T-SPOT results were 0.07 and 0.19 when using the fixed effect model and 0.08 and 0.10 using the random effects model, respectively. Overall, the QFT-IT assay appeared to reduce the occurrence of indeterminate results among HIV-seropositive individuals. This conclusion seems more meaningful for diagnosis of LTBI, because an indeterminate result is the major limitation of the IGRA inimmunosuppressed subjects. The reason for the greater ratio of indeterminate results in the case of the T-SPOT might be associated with technical error due to the more sophisticated technical demand compared with the QFT-IT assay .2 = 94.48 and 97.92 % for sensitivity and specificity) and the small number of included studies, the results may not accurately represent the performance of the T-SPOT assay. Furthermore, more additional trials are required to determine the efficiency of the T-SPOT assay.The SROC curve for the T-SPOT assay was positioned in the upper left corner, and the AUC was 0.93 , which may suggest a more effective performance for distinguishing ATB cases among HIV-infected individuals than the QFT-IT assay, which produced an unsatisfactory AUC of 0.78 . However, as evidenced by the high heterogeneity among the studies (IP = 0.04) for the T-SPOT assay in our meta-analysis, as shown in Fig. P = 0.47), which suggested a low risk of publication bias.The potential risk of publication bias was significant (Our systematic review also has some limitations. Due to our strict selection and exclusion process, the number of included studies was only 11. Moreover, there was heterogeneity among the studies included in our analysis, and the difference between low/intermediate-burden countries and high-burden countries may be one of the reasons for the heterogeneity.In summary, the latest evidence provided in this systematic review and meta-analysis shows the current limited ability and accuracy of the IGRA for diagnosing ATB and ruling out non-ATB cases; therefore, the T-SPOT assay may be more accurate for distinguishing ATB cases among HIV-infected individuals, while the QFT-IT assay appears to reduce the occurrence of indeterminate results. Thus, the IGRA does not appear to be optimal for the clinical confirmation of ATB cases in HIV-seropositive individuals until its efficiency is verified, although modifications and additional trials may help to improve its effectiveness.95 % CI, 95 % confidence intervals; AIDS, acquired immunodeficiency syndrome; ATB, active tuberculosis; HIV, human immunodeficiency virus; IFN-γ, interferon-γ; IGRA, interferon-γ release assay; IQRs, interquartile ranges; LTBI, latent tuberculosis infection; PRISMA, preferred reporting items for systematic reviews and meta-analyses; QFT-IT, quantiFERON-TB gold in-tube assay; QUADAS-2, quality assessment of diagnostic accuracy studies-2; SROC, symmetric receiver operator characteristic; TB, tuberculosis; T-SPOT, T-SPOT.TB assay; WHO, world health organization


Our results show that maximal force was not significantly different between KO and WT preparations but length-dependent increase in pCa50 was attenuated in the KO preparations. pCa50 was not significantly different between WT and KO preparations at long SL (5.82 ± 0.02 in WT vs. 5.87 ± 0.02 in KO), whereas pCa50 was significantly different between WT and KO preparations at short SL . The ktr, measured at half-maximal Ca2+-activation, was significantly accelerated at short SL in WT preparations . Furthermore, krel and kdf were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, ktr was not altered by changes in SL in KO preparations (8.03 ± 0.54 s−1 at 1.9 μm vs. 8.90 ± 0.37 s−1 at 2.1 μm). Similarly, KO preparations did not exhibit length-dependent changes in krel and kdf. Collectively, our data implicate cMyBP-C as an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL.Enhanced cardiac contractile function with increased sarcomere length (SL) is, in part, mediated by a decrease in the radial distance between myosin heads and actin. The radial disposition of myosin heads relative to actin is modulated by cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C contributes to the length-dependent activation (LDA) in the myocardium. However, the precise roles of cMyBP-C in modulating cardiac LDA are unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca Our results show that the length-dependent increase in pCa50 was attenuated in the KO preparations compared to wild-type (WT) preparations. Furthermore, length-dependent changes in dynamic contractile parameters ktr, krel, and kdf were blunted in KO preparations compared to WT preparations, indicating that cMyBP-C plays a critical role in the myofilament-mediated response in cardiac LDA.Importantly, recent evidence from low-angle X-ray diffraction experiments showed that cMyBP-C tethers the myosin XBs closer to the thick-filament backbone and that ablation of cMyBP-C results in the radial displacement of XBs closer to the thin-filament , and was conducted according to the guidelines of the Institutional Animal Care and Use Committee at Case Western Reserve University. Mice aged 3–6 months, of both sexes, and belonging to SV/129 strain were used for the experiments. KO mice used in this study were previously generated and well-characterized  also contained 7 EGTA and 7.01 CaCl2; while the relaxing solution (pCa 9.0) contained 7 EGTA and 0.02 CaCl2; and the pre-activating solution contained 0.07 EGTA. The pH of the Ca2+ solutions was set to 7.0 with KOH and the ionic strength was 180 mM. A range of pCa solutions, containing varying amounts of [Ca2+]free, were then prepared by mixing appropriate volumes of pCa 9.0 and 4.5 stock solutions and the experiments were performed at 22°C.Skinned ventricular multicellular preparations were prepared as described previously  i.e., P/Po to construct the force-pCa relationships  of the sarcomeres in the heart muscle , where Hn is the Hill coefficient and k is the pCa required to produce half-maximal activation   and a force transducer  as described previously , and once the muscle preparations attained steady-state isometric force, they were rapidly slackened by 20% of their original muscle length and were held for 10 ms using a high-speed length control device (Aurora Scientific Inc.). The slackening was followed by the brief period of unloaded shortening which resulted in a rapid decline in force due to detachment of the strongly-bound XBs. The muscle preparation was then rapidly restretched back to its original length and the time course of force redevelopment was measured. ktr for each slack-restretch maneuver was estimated by linear transformation of the half-time of force redevelopment, i.e., ktr = 0.693/t1/2, as described previously , with a characteristic rate constant kdf, which is a result of length-induced recruitment of new XBs into the force-bearing state  comparisons.Data were analyzed using Two-Way analysis of variance (ANOVA). One factor in this analysis was cMyBP-C variant (WT or KO), and the second was SL (1.9 or 2.1 μm). Therefore, we used Two-Way ANOVA to test the hypothesis that the effect of the SL on a given contractile parameter depended on the cMyBP-C variant (interaction effect). When the interaction effect was significant, it showed that the effects of SL on various contractile parameters were different in the presence or absence of cMyBP-C. When the interaction effect was not significant, we interpreted the main effect due to cMyBP-C variant or SL. Planned multiple pairwise comparisons were made using Fisher's LSD method  was measured at SL's 1.9 and 2.1 μm in WT and KO muscle preparations  of SL on Ca2+-activated maximal force production. To probe the determining factor for the significant main effect, subsequent post-hoc tests were carried out. These post-hoc tests using multiple planned pairwise comparisons showed that maximal force production was not significantly different between WT and KO groups at either SL (Table max) was significantly decreased by ~34% and ~38% at short SL vs. long SL in WT and KO groups, respectively (Table 2+-independent forces measured at pCa 9.0 (Fmin) in WT and KO groups  and cMyBP-C (P < 0.005) on pCa50. Subsequent post-hoc tests revealed that the main effect of SL was because of the following: pCa50 significantly increased upon increasing the SL from 1.9 to 2.1 μm as indicated by a leftward shift in the force-pCa relationships in both WT and KO groups  was attenuated in KO group when compared to ΔpCa50 of WT group  on nH suggesting that cMyBP-C influenced the effect of SL on nH. Subsequent post-hoc tests revealed that nH significantly increased by ~38% at SL 1.9 μm in WT group, a result that agrees with earlier studies  on ktr suggesting that cMyBP-C influenced the effect of SL on ktr. The cause of the interaction effects was assessed by post-hoc multiple pairwise comparisons which showed that submaximal ktr was accelerated in KO compared to WT group at long SL  + g (rate of XB detachment) according to a two-state XB model  to measure krel and kdf which are measures of the rates of XB detachment and XB recruitment, respectively  on krel suggesting that cMyBP-C influenced the effect of SL on the rate of XB detachment kinetics. The cause of the interaction effect was evident from the post-hoc multiple pairwise comparisons which revealed that krel was significantly accelerated by ~32% at short SL in WT group but such an acceleration of krel at short SL was absent in KO group  on kdf suggesting that cMyBP-C influenced the effect of SL on the rate of XB recruitment into the force-bearing state. The cause of the interaction effect was assessed using post-hoc tests which revealed that kdf was significantly accelerated by ~50% at short SL in WT but such an acceleration of kdf at short SL was absent in KO  was accelerated at short SL compared to long SL in WT group  at short SL compared to long SL in WT group  at long SL in KO compared to WT  upon an increase in SL is a hallmark of LDA  . In this study we found a similar trend, although here the ΔpCa50 was 0.11pCa units in WT skinned myocardium whereas it was 0.07 pCa units in KO skinned myocardium  via changes in the interaction between tropomyosin (Tm) and TnI  and detachment (g)  and force decay (krel), parameters that are analogous to f and g. Our data shows that acceleration of ktr at short SL in WT group was indeed due to a combination of increases in both kdf and krel (Figure kdf and krel did not increase at short SL in KO skinned myocardium such that the values were not significantly different from those at long SL (Figure ktr at short SL in KO group was due to the absence of accelerations in both kdf and krel at short SL (Figure Because l Figure . Our datL Figure . A recenL Figure  showed tL Figure . Thus, okrel, Figure krel (Stelzer et al., krel seen in KO group (Figure To test whether changes in XB detachment (as assessed by p Figure  may havep Figure , becausekdf, Figure nH from the pCa-tension relationships. Our estimates showed that nH values were increased in WT at short SL compared to long SL (Figure nH may be a result of enhanced Ca2+ binding to Tn, near-neighbor interactions among Tn-Tn, XB-Tn, and XB-XB (Razumova et al., nH at short SL was absent in KO (Figure kdf in the KO group (Figure 2+/TnC (Li et al., nH with decrease in SL. Therefore, our data shows that length-dependent changes in cooperative mechanisms are depressed when cMyBP-C is absent in the sarcomere.To test whether changes in XB recruitment, (as assessed by L Figure , a resulL Figure . This suO Figure  which map Figure . In the p Figure , XB-Tn (p Figure  and XB-C2+, XB cycling kinetics, and muscle fiber stiffness. Taken together, our findings suggest that impaired LDA may contribute to depressed myocardial contractile function in human patients harboring mutations in cMyBP-C that ultimately cause a significant decrease in the amount of cMyBP-C expression in the sarcomere.Our study provides evidence to show that cMyBP-C plays a key role in fine-tuning length-dependent cardiac contractile function via its impact on myofilament responsiveness to CaRanganath Mamidi and Julian E. Stelzer contributed to the conception and design of the experiments. Ranganath Mamidi, Kenneth S. Gresham, and Julian E. Stelzer participated in performing the experiments, data acquisition, data analysis, data interpretation, drafting, and revising the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


APOA5) genetic variants and hypertriglyceridemia has been extensively studied, there have been few studies, particularly in children and adolescents, on the association between APOA5 genetic variants and obesity or non-high-density lipoprotein cholesterol (non-HDL-C) levels. The objective of this study was to examine whether APOA5 gene polymorphisms affect body mass index (BMI) or plasma non-HDL-C levels in Chinese child population.Although the association between the apolipoprotein A5 (This was a case–control study. Single nucleotide polymorphisms (SNPs) were genotyped using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry for an association study in 569 obese or overweight and 194 healthy Chinese children and adolescents.APOA5 gene were all increased in obese or overweight patients compared to the controls. After adjusted for age and sex, C carriers in rs662799 had a 1.496-fold  higher risk for developing obesity or overweight than subjects with TT genotype, while C carriers in rs651821 had a 1.515-fold higher risk than subjects with TT genotype . Triglyceride (TG) and non-HDL-C concentrations were significantly different among rs662799 variants and both were higher in carriers of minor allele than in noncarriers for TG (1.64 ± 0.96 vs. 1.33 ± 0.67 mmol/L) (P < 0.001), and for non-HDL-C (3.23 ± 0.92 vs. 3.02 ± 0.80 mmol/L) (P = 0.005), respectively. There was also a trend towards increased TG and non-high-density lipoprotein cholesterol levels for rs651821 C carriers . Furthermore, to confirm the independence of the associations between APOA5 gene and TG or non-HDL-C levels, multiple linear regression analysis was performed and the relationships were not eliminated by adjustment for age, sex and BMI.Genotype distributions for all polymorphisms in both cohorts were in accordance with the Hardy-Weinberg distribution. The frequencies of the risk alleles in rs662799 and rs651821 SNPs in APOA5 gene may influence the susceptibility of the individual to obesity, which may also contribute to an increased risk of high non-HDL-C levels in Chinese obese children and adolescents.These findings suggest the TG-raising genetic variants in the  Obesity is one of the most complex clinical syndromes facing children, almost one-third of obese children remain obese in adulthood and that the risk of obesity as an adult was close to twice as high for obese children compared with those who had the normal weight during the growing period . One of APOA5) gene, located on chromosome 11 adjacent to APOA1/APOC3/APOA4 gene cluster with known functions in the metabolism of plasma lipids  than subjects with TT genotype, while carriers of the C allele (CC + TC) in rs651821 had a 1.486-fold higher risk than subjects with TT genotype . These associations remained significant after controlling for age and sex .We then employed logistic regression to test the effect of the two APOA5 variants and obesity-related traits in obese or overweight children were examined. Figure APOA5 polymorphism and TG levels in Chinese children and adolescents. Also, non-HDL-C was influenced significantly by APOA5 gene variants. We noted that the non-HDL-C in obese or overweight children with non-TT (CC + TC) genotype was 3.23 ± 0.92 mmol/L, which was elevated than that in obese children with TT genotype in rs662799 (3.02 ± 0.80 mmol/L) with a significant difference (P = 0.005). There was also a significant trend towards increased non-HDL-C levels for APOA5 rs651821 C carriers .Next, the association between APOA5 gene polymorphisms and TG or non-HDL-C levels, multiple linear regression models, using transformed log-TG as the dependent variable and including both APOA5 rs662799 and rs651821 variants and other lipid influencing factors  as the covariates, were performed in obese or overweight children. Carriership for APOA5 rs662799 risk allele was identified as a significant and independent predictor of both TG  and non-HDL-C variabilities . Similarly, carriership for APOA5 rs661821 risk allele was a significant and independent predictor for both TG  and non-HDL-C variabilities   confers susceptibility to development of obesity in pediatric patients [APOA5, however, little is known about the associations between APOA5 variants and body fat composition. Thus, mechanisms underlying our findings still remain hypothetic, requiring further work to prove. One speculated mechanism is that the effect of APOA5 gene variants on the risk of obesity might be attributed to this locus being in interaction with other obesity-risk genes, such as APOA1, APOC3 and APOA4 genes [APOA1/C3/A4/A5 cluster occupies a restricted chromosomal region, phenotypic effects that appear to result from mutations in one member of the cluster might actually derive from allelic associations with functional variants in another [APOA5 variants, resulting in varying degrees of impaired lipolysis [APOA5 gene variants, as a result, further studies are needed to clarify this issue.Our available data supported the hypothesis that the TG-raising genetic variants in the patients . Additiopatients . Until nA4 genes ,16. Sinc another . Other pipolysis . As obesAPOA5 genetic variants and hypertriglyceridemia has been extensively studied [APOA5 genetic variants and non-HDL-C levels. Furthermore, the relationships for TG or non-HDL-C were not eliminated by adjustment for age, sex and BMI, suggesting that this genetic factors act independently of age, sex or BMI. In consistent with our results, Hubacek et al. also suggested that the rs662799 variant in the APOA5 gene could be a significant genetic determinant for plasma non-HDL-C levels [APOA5 gene variants could affect non-HDL-C levels. It is known that APOA5 protein accelerates plasma hydrolysis of TG-rich lipoproteins by activation of plasma LPL [Although the association between  studied -21 and aC levels . So far asma LPL . We specThe current study has several limitations that should be noted. First, the interactions between gene-gene, gene-environment and even different polymorphic loci of the same gene may influence the biological effects of the polymorphisms of the genes. Second, for all the polymorphisms, the number of the available studies in some subgroups, such as the control group was small. Third, due to the cross-sectional nature of the study design, the causal relationships between gene variants and clinical disorders can’t be drawn. Thus, we hope to perform full longitudinal association analyses in larger samples and more candidate genes in the future prospective studies. Furthermore, studies in the other populations will be useful to confirm our results.APOA5 gene may influence the susceptibility of the individual to obesity, which may also contribute to an increased risk of high non-HDL-C levels in Chinese obese children and adolescents. We believe that our findings will improve the understanding of biological functions of APOA5 gene variants.In conclusion, we herein demonstrated the TG-raising genetic variants in the This case–control study was conducted from 2011to 2012. We totally recruited 569 obese or overweight patients (“case”) at the age of 7–16 from outpatients who visited one of the following three medical centers for their weight problems: (1) Department of Endocrinology, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang; (2) Department of Child Health Care, the Affiliated Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang; (3) Department of Child Health Care, Ningbo Women & Children’s Hospital, Ningbo, Zhejiang. According to the diagnostic criteria developed by the Working Group for Obesity Task Force in China , the 85tInformed consent was obtained from the parents, and approved by the ethics committee of all the aforementioned three medical centers: the Children Hospital of Zhejiang University School of Medicine, the Affiliated Yuying Children’s Hospital of Wenzhou Medical University and Ningbo Women & Children’s Hospital.2). Age- and sex-specific BMI z-scores were used as continuous dependant variables for each model [Body height was measured to the nearest 0.1 cm, while weight was measured to the nearest 0.1 kg. At the time the measurements were made, the participants were only their underwear. BMI was calculated as body weight (kg) divided by the square of body height . Non-HDL-C was calculated as TC level minus HDL-C. The remaining blood samples were stored at -80 degrees Celsius for SNP genotyping.DNA was isolated from blood samples using Blood Genomic DNA Miniprep Kit (AxyPrep) according to the protocol of the manufacturer. All samples were originally genotyped using an automated platform MassARRAY .DNA sequence containing the target SNP site was first amplified by polymerase chain reaction. By using the SNP specific primers, the products were extended one base in SNP sites. After applied into the MassARRAY SpectroCHIP array, the products were crystallized with matrix in the chip. And then the crystal containing chip was directed into the mass spectrometer vacuum tube and excited using an instantaneous nanosecond (10–9 s) laser. The molecular of matrix absorb the laser radiation, which resulted in energy accumulation causing crystal matrix sublimation, DNA molecule desorption and transformation to metastable ions .t-test. The chi-square test was employed to test for deviations of the genotypes from the Hardy-Weinberg equilibrium and to evaluate categorical variables. To determine whether SNP polymorphisms were independent modulators on the development of obesity, logistic regression analysis was performed adjusted for age and sex. We applied ANOVA and the Studens’s t test to compare crude means across genotype groups. A multiple linear regression models procedure was performed by using transformed log-TG as the dependent variable, and including carrierships of APOA5 variants and other influencing factors  as the covariates. The power calculation was performed using Quanto software (http://hydra.usc.edu/gxe/). Our study had ≥68.1% to ≥98.5% power (enrolment of 569 cases and 194 controls) to detect Ors of 1.50 to 2.00 for obesity under an additive model, assuming a significance of 0.05, an allele frequency of 0.267, and overweight or obesity prevalence of 19.9% in 7- to 16- year old Chinese Children [P-value <0.05 was considered statistically significant.Quantitative variables were expressed as mean ± standard deviation (SD) and differences between groups were calculated by Student’s Children . Statistnon-HDL-C: Non-high-density lipoprotein cholesterol; TC: Total cholesterol; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; APOA5: Apolipoprotein A5; BMI: Body mass index; TG: Triglyceride; OR: Odd ratio; CI: Confidence interval; LPL: Lipoprotein lipase.The authors have no competing interests.Dr. WZ did the experiments and data analysis, and drafted the initial manuscript. Prof. LL designed thestudy, and critically reviewed and revised the manuscript. Dr. CW and Prof. JF also designed the study and participated in critical discussion. Dr. ZS mainly collected the data and participated in doing experiments. Dr. PL and Dr. LL both all contributed to the collection of data. Prof. YZ provided help for the data analysis and interpretation. All authors read and approved the final manuscript.We all agree that every author is the co-first author of the paper.


Experiment 2 investigated performance in the same participants when instructed to feign symptoms of traumatic brain injury (TBI): 87% produced abnormal omnibus z-scores. Simulated malingerers showed greater elevations in simple reaction times than CRTs, and hence reduced CentPTs. Latency-consistency z-scores, based on the difference between the CRTs obtained and those predicted based on CentPT latencies, discriminated malingering participants from controls with high sensitivity and specificity. Experiment 3 investigated CRT test performance in military veterans who had suffered combat-related TBI and symptoms of post-traumatic stress disorder, and revealed small but significant deficits in performance in the TBI population. The results indicate that the new CRT test shows high test–retest reliability, can assist in detecting participants performing with suboptimal effort, and is sensitive to the effects of TBI on the speed and accuracy of visual processing.Choice reaction time (CRT), the time required to discriminate and respond appropriately to different stimuli, is a basic measure of attention and processing speed. Here, we describe the reliability and clinical sensitivity of a new CRT test that presents lateralized visual stimuli and adaptively adjusts stimulus onset asynchronies using a staircase procedure. Experiment 1 investigated the test–retest reliability in three test sessions performed at weekly intervals. Performance in the first test session was accurately predicted from age and computer-use regression functions obtained in a previously studied normative cohort. Central processing time (CentPT), the difference between the CRTs and simple reaction time latencies measured in a separate experiment, accounted for 55% of CRT latency and more than 85% of CRT latency variance. Performance improved significantly across the three test sessions. High intraclass correlation coefficients were seen for CRTs (0.90), CentPTs (0.87), and an omnibus performance measure (0.81) that combined CRT and minimal SOA  Choice reaction times (CRTs) have been widely used to quantify attention and processing speed in clinical populations, including patients with head injury , post-trFigure 1). Stimulus onset asynchronies (SOAs) are adjusted adaptively based on participant performance and stimuli are delivered randomly to the left or right hemifield to evaluate lateralized processing deficits.The CRT test incorporates a rapid serial visual feature-conjunction task in which participants press one mouse button in response to a target letter (blue P) and press the other mouse button to respond to non-target letters that share color (blue F), shape (orange P), or neither target feature  at the Veterans Affairs Northern California Health Care System (VANCHCS) and were compensated for their participation. Participants underwent three test sessions at weekly intervals.The demographic characteristics of the participants in Experiment 1 are shown in lsewhere . The 46 1 Testing was performed in a quiet room using a standard personal computer (PC) controlled by Presentation® software .Choice reaction time testing required 4–6 min per participant and occurred after simple reaction time (SRT) testing, mid-way through a battery of cognitive tests.Figure 1 shows the paradigm.2 Participants responded to the target  by pressing the left mouse button, and responded to the three different non-target stimuli (probability 20% each) by pressing the right mouse button. The response buttons were reversed for left-handed participants. The letters P and F appeared in blue or orange colors , with non-target stimuli differing from the target in color (orange P), shape (blue F), or both features (orange P). Stimulus durations were fixed at 200 ms. Participants were trained to criterion levels (80% correct) in 20 practice trials, repeated if necessary, and then performed 140 test trials. SOAs were initially set at 2500 ms and were reduced by 3% following two successive correct responses and increased by 3% following each error or response omission.Hardware calibrations showed a delay of 11.0 ± 0.1 ms in the illumination of the 17” Samsung Syncmaster monitor when measured with a photodiode. Delays associated with the high-speed computer-gaming mouse  were measured with an electronic relay and showed a mean of 6.8 ± 1.8 ms. Thus, mean hardware delays totaled 17.8 ms.0, the last reported time before the event occurred; T1, the time of the event-monitoring loop associated with event occurrence; and T2, the time following the timing loop in which the event occurrence was first noted. While the event may have occurred before the time T1, Presentation may have read the time (T1) before testing for the response in the loop cycle. As a result, the time T2 must be used to define the upper limit of event latency. For example, consider the case where Presentation determined that no response was evident in a loop at 480.0 ms (T0), a loop at 480.1 ms (T1) was associated with a response, and a loop at 480.2 ms (T2) occurred after the response. These times establish a time range, T2-T0, which includes a time, T0 (480.0 ms), that was definitely before the response, and a time, T2 (480.2 ms), that was definitely after the response. Therefore, Presentation reports the response latency as T0 (480.0 ms) with an associated time uncertainty of T2-T0 (0.2 ms).Presentation software reports timing-uncertainty measures for each event to quantify the timing precision as tests are performed. The occurrence of each event is associated with three different event times measured with the high-precision (100 kHz) programmable clock: T2-T0 would be increased by 10 ms.While Presentation software minimizes interruptions due to disk access and computational operations intrinsic to the test by interleaving these operations with the high-speed event-timing loop, timing errors can still occur if the event-timing loop is interrupted by resource-demanding operations  or operating system processes. For example, if a 10 ms operating system interruption occurred at the instant that the subject responded in the trial described above, the CRT latency reported would be unchanged (480.0 ms), but the event-time uncertainty TThe event-time uncertainties reported by Presentation for 10,379 stimulus events in Experiment 1 showed a mean of 0.12 ms (±0.34), with three stimuli showing event-time uncertainties greater than 1.0 ms, including one instance where stimulus-time uncertainty was 18.2 ms. The time uncertainties reported for 7,353 response events showed a mean of 0.24 ms (±0.67), with 17 responses showing timing uncertainties greater than 1.0 ms, including one instance where response-time uncertainty was 41.5 ms. In summary, these results indicate that software timing was extremely precise, and software timing errors had minimal influence on the results.We quantified mean CRTs along with intrasubject  CRT standard deviations and hit rates for each type of stimulus. A response window of 250–1250 ms was used: failure to generate a response during this interval was categorized as a miss, and an incorrect response occurring within the window was categorized as an error. The minimum SOA (mSOA), which reflected the ability of participants to accurately respond to stimuli presented at rapid rates, was also measured for each participant. In cases where SOAs were reduced below 1250 ms, multiple responses could occur within a response window. In these cases, responses were assigned to stimuli in the order in which they occurred.Overall performance was examined using mean CRTs and central processing time (CentPT), the difference between CRTs and SRTs measured in a companion test performed on the same day . In addir = -0.15, t(1464) = 4.80, p < 0.0001]. Multiple regression analysis showed that the combined influence of age and computer-use accounted for 22.5% of CRT variance, with both factors exerting significant effects . Age and computer-use also had significant effects on CentPT latencies . Therefore, CRT and CentPT z-scores in the current study were calculated after regressing out the effects of age and computer-use on the observed CRT and CentPT latencies. In contrast, age and computer-use had little influence on log-mSOA measures, where they accounted for less than 1% of the variance. Therefore, no regressions were used when calculating log-mSOA z-scores. Finally, omnibus z-scores were used to summarize overall performance by adding CRT z-scores and log-mSOA z-scores, and then normalizing the resulting distribution.Previous large-scale studies showed a large effect of age on performance, but minimal effects of education . The reawww.ebire.org/hcnlab). Greenhouse–Geisser corrections of degrees of freedom were uniformly used in computing p-values in order to correct for covariation among factors and interactions. Effect sizes are reported as Cohen’s d values or as t-test. ICCs were calculated with SPSS .The results were analyzed with analysis of variance (ANOVA) using CLEAVE , along with the results from the normative control population (blue diamonds). Figure 3 shows a similar plot of mSOAs. Figure 4 shows CentPTs as a function of age, and Figure 5 shows the relationship between CRT and omnibus z-scores in the individual participants, after regressing out the effects of age and computer-use. CRTs in Experiment 1A averaged 494 ms, and mSOAs averaged 766 ms. A summary of the different measures is provided in Table 2.Mean CRT latencies from the individual participants in the first test session of Experiment 1 (1A) are shown as a function of age in z-scores [-0.01] in Experiment 1A were virtually identical to those predicted by the age-regression functions from F = 0.01, NS]. Neither CentPT z-scores  nor omnibus z-scores  differed significantly from those in the normative group. However, there was a trend toward lower log-mSOA z-scores .Age- and computer-use regressed CRT Figure 6 shows comparisons of CRT latencies across the three test sessions. ICCs across the three test sessions were used to evaluate test–retest reliability. ICCs were high for CRT z-scores (0.89), CentPT z-scores (0.86), and omnibus z-scores (0.81), while they were somewhat lower for log-mSOA z-scores (0.72), and considerably reduced for measures of trial-by-trial CRT variance (0.41) and the CV (0.27).Figure 1), and Test Session . There was a significant effect of Stimulus-Type on CRT latencies .There was also a significant interaction between Test Session and Stimulus-Type , presumably because SOAs were reduced as performance improved, nor did Sessions interact significantly with the effect of Stimulus-Type on hit rate . However, there was a significant reduction in the magnitude of the Spatial Compatibility effect on hit rate over repeated sessions . Because CentPTs were reduced in simulated malingerers, predicted CRT latencies based on CentPT latencies (381 ms) were much shorter than those actually observed (609 ms). As a result, the mean latency-consistency z-score in simulated malingerers was 8.64, and, as shown in Figure 8, all but one of the simulated malingerers produced latency-consistency z-scores outside the normal (p < 0.05) range. With a more conservative latency-consistency z-score cutoff of 3.0, 87% of simulated malingerers showed abnormalities. In contrast, less than 1% of normal controls produced latency-consistency z-scores > 3.0, including fewer than 4% of control participants with abnormal Omnibus z-scores.Simulated malingerers showed greater slowing in the SRT than the CRT task, resulting in reductions in CentPTs . These abnormalities in latency-consistency z-scores reflected the inability of simulated malingerers to accurately adjust their performance “deficits” in SRT and CRT tests.A latency-consistency A number of previous studies have suggested that malingering effects are often reduced as task complexity increases. For example, Task-complexity effects in malingering participants may reflect the dual-task nature of malingering: malingerers must perform a primary task  while simultaneously monitoring and adjusting performance to simulate impairment. As test difficulty increases, fewer cognitive resources are available to monitor and adjust performance. As a result, the relative magnitude of the malingering deficit decreases. For example, malingering effects are larger in the Trail Making Test, Part A than on the Trail Making Test, Part B . We founThe participants in Experiment 2 had undergone three prior CRT test sessions before participating in the malingering experiment. Since repeated testing resulted in improvements in CRT performance, malingering effects would be somewhat larger when compared to “baseline” performance in session 3 than in session 1.Traumatic brain injury can result in impairments in attention and processing speed . For exaTable 1) who had previously been evaluated with other computerized tests , with an average 13.6 years of education , with a maximal uncertainty of 0.9 ms. Uncertainties for 3,559 responses averaged 0.17 ms (±0.09 ms), with a maximal uncertainty of 0.9 ms.Results from Experiment 3 were compared to the normative population data using the age and computer-use regression functions established in Table 2. In comparison with the normative controls, omnibus z-scores were increased in the TBI patient group , but increases in log-mSOA z-scores showed only a trend . A comparison of the TBI group with the participants in Experiment 1A showed increases in both omnibus z-scores  or in comparison with Experiment 1A participants . CentPT z-scores were also similar in the TBI patients and controls . As in controls, CentPTs in the TBI patients correlated very strongly with CRTs (r = 0.96).A summary of the results of Experiment 3 is included in Stimulus-type and compatibility effects did not differ significantly from those seen in control participants; ANOVAs with Group (TBI patients vs. normative controls), Stimulus-type, and Stimulus-Response Spatial-Compatibility as factors showed no significant Group differences in Stimulus-Type or Spatial-Compatibility for either CRT or hit rate measures in comparisons with either the normative control group or the participants in Experiment 1A.z-scores  and Experiment 1A participants .Omnibus z-scores, largely reflecting increases in log-mSOA z-scores. Increases in CRT z-scores were also evident, but only trended toward significance. We found no differences in the effects of Stimulus-Type and Stimulus-Response Spatial Compatibility in TBI patients and controls. This suggests that TBI patients experienced the same relative increase in difficulty with harder-to-discriminate distractors and stimulus-response incompatibility as controls. Significant omnibus z-score elevations were seen in five TBI patients, all of whom had latency-consistency z-scores within the normal range. This pattern was opposite to that seen in the simulated malingerers, who generally showed much larger increases in latency-consistency z-scores than omnibus z-scores.The TBI patient group showed small but significant impairments in omnibus z-score > 3.0 cutoff developed in Experiment 2 correctly assigned 96% of TBI patients to the non-malingering group. The latency-consistency metric developed in Experiment 2 also detected two mTBI patients identified in previous studies as performing with suboptimal effort. Both of these patients showed large elevations in omnibus z-scores (4.89 and 3.02). Had these patients been included in the mTBI patient group, group-level differences with control populations would have been substantially enlarged. This highlights the importance of incorporating performance-validity metrics in studies of TBI patient populations  or organic causes  with 87% sensitivity and 96% specificity. This suggests that the CRT test not only provides useful information about processing speed and attention, but is also a sensitive performance-validity test that can detect suboptimal effort.The CRT test was found to be sensitive in detecting simulated malingerers based on inconsistencies between CentPT and CRT measures. In particular, a latency-consistency z-score (0.81) performance measures, which evaluated both processing speed and accuracy. Performance improved significantly over three test sessions. Experiment 2 studied the same participants when instructed to feign symptoms of TBI. More than 87% of the simulated malingerers showed abnormal performance. A latency-consistency z-score metric accurately discriminated simulated malingerers from controls with 87% sensitivity and 99% specificity, including 96% specificity when considering only controls with abnormal performance. Experiment 3 found small but significant performance deficits in 26 military veterans with TBI that were more severe in patients who had suffered severe rather than mild TBI. The CRT test reveals the effects of learning, simulated malingering, and TBI on the speed and accuracy of visual stimulus processing.Visual CRTs in a rapid serial visual feature-conjunction test were studied in three experiments. Experiment 1 investigated the effects of repeated testing in a highly educated, young control group whose initial performance was well-predicted by regression functions obtained in a normative population of control participants. ICCs were high for both CRT (0.90) and omnibus David L. Woods is affiliated with NeuroBehavioral Systems, Inc., the developers of Presentation software that was used in these experiments. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


First, the liposome tethering properties of the Golgi resembles that of the amphipathic lipid-packing sensor (ALPS) motif of GMAP-210: both preferred small (radius < 40 nm) liposomes made of monounsaturated but not saturated lipids. Second, reducing GMAP-210 levels or redirecting its ALPS motif to mitochondria decreased liposome capture by the Golgi. Extensive mutagenesis analysis suggests that GMAP-210 tethers authentic transport vesicles via the same mechanism whereby the ALPS motif senses lipid-packing defects at the vesicle surface through its regularly spaced hydrophobic residues. We conclude that the Golgi uses GMAP-210 as a filter to select transport vesicles according to their size and bulk lipid composition.When small phosphatidylcholine liposomes are added to perforated cells, they bind preferentially to the Golgi suggesting an exceptional avidity of this organelle for curved membranes without stereospecific interactions. We show that the DOI:http://dx.doi.org/10.7554/eLife.16988.001 Exchange of material between organelles is largely mediated by transport vesicles, which bud from the surface of a donor compartment through the mechanical action of protein coats and fuse with an acceptor compartment through the action of SNARE proteins . Betweencis side of the Golgi preferentially capture vesicles trafficking between the endoplasmic reticulum (ER) and the cis Golgi, whereas those that are normally anchored at the trans side preferentially capture vesicles from endosomal sources. Although the molecular determinants responsible for this selectivity are not well known, discrete binding sites for Rab proteins have been identified along the coiled-coils of golgins, suggesting a mechanism by which vesicles move within the golgin matrix by transient Rab-coiled-coil contacts  rather than a trans (TGN46) localization , inserting two alanines to break the regular spacing of polar and non-polar residues (InsAA20), and replacing a glycine by a proline to create a bend (G11P). In all cases, the fraction .10.7554/The ALPS motif of GMAP-210 displays a monotonous sequence made of similar helical turns  in their polar face and contain bulky hydrophobic residues  in their non-polar face . As compLxxxLxxxL); in Arf1 and Sar1, most helical turns contain two adjacent hydrophobic residues (e.g. IFxxLFxxLF in Arf1). To determine whether the sparse distribution of hydrophobic residues in ALPS plays a role in membrane selectivity, we created a condensed ALPS motif (condALPS) by deleting polar residues such as to create hydrophobic pairs akin to what is found in Arf or Sar . For this, we built coarse-grained models of bilayers of different shape with a composition approaching that of the ER/cis Golgi interface  were cand 40 nm .cis entrance of the Golgi to select transport vesicles according to their size and bulk lipid composition.The Kobayashi and Pagano experiment was remarkable in its simplicity – mixing liposomes with perforated cells – and in its outcomes: the Golgi has an exceptional liposome tethering capacity, which is accompanied by size filtration . By connca. 400 vesicles in a tomogram corresponding to 5% of the Golgi area, whereas Marsh et al. reported >2000 vesicles in a larger region  suggested a mechanism for vesicle capture , 2008. Tr region . Other cr region  as well r region . Howeverr region , we idenr region . This mor region .Mitochondria de-routing experiments indicate that GMAP-210 is selective for vesicles of the early secretory pathway . FurtherALPS motifs differ from most membrane-adsorbing helices by the lack of charged residues and the abundance of small neutral residues in the polar face . The precis Golgi differ from that of other organelles in their bulk features, including low sterol, low electrostatics and high level of unsaturation  occur mostly at the trans side  have been previously described . The ALPFor ALPS-ACC1-GFP-FKBP, we inserted ALPS-ACC1-GFP in a ProteoTuner vector  that contains a modified FKBP domain (degradation domain). This domain was mutated back to the wild-type form by introducing the following mutations: G31E, V36F, G71Q and E105K. Mito-FRB was a gift from M. Lowe .2 humidified atmosphere at 37°C. hTERT-RPE1 cells stably expressing Rab6-GFP (gift from B Goud) or ArfGAP1(R50K)  were cultured in DMEM/F12 medium with glutaMAX  containing 10% serum, 1% antibiotics  and were incubated in a 5% COP1(R50K)  were culP1(R50K) . PurifieFor lipid droplets staining, live cells were incubated for 10 min with HCS lipidTOXred neutral lipid stains (Invitrogen). Experiments with live cells were performed using a spinning disk confocal microscope .2), blotted against filter paper, and placed on top of the coverslip. After 1 min at room temperature, the filter was slowly peeled from the cells. The coverslip was placed in a support for life microscopy, and 400 µl of liposomes solution was immediately added. After 5 min of incubation at 30°C, cells were washed several times with DMEM/F12 medium without red phenol (Gibco) and cells were observed in a wide-field microscope (Olympus IX83). The Golgi area was determined by the Rab6-GFP signal, and a corresponding mask was then applied to the red channel to determine the liposome fluorescence in the same area.We followed the method previously described . hTERT-RCells were transfected with 15 nM siRNA using Lipofectamine RNAiMAX  for a 35 mm glass coverslip. ON-TARGET plus siRNAs targeting GMAP-210 were from Dharmacon . They correspond to the siRNAs used by Roboti et al.  and havesiGMAP-210#1: GGAGAUAGCAUCAUCAGUA,ref:J-012684-05-0002siGMAP-210#2: CAAGAACAGUUGAAUGUAG, ref:J-012684-06-0002non-targeting siRNA: UGGUUUACAUGUUGUGUGA, ref:D-001810-02-05After 72 hr of transfection, cells were used for western blotting or for perforation. Expression of GMAP-210 was revealed with a purified mouse anti-GMAP-210 antibody  and the loading control was revealed with a monoclonal mouse anti-GFP antibody .Cells coexpressing ALPS-ACC1-GFP-FKBP and Mito-FRB were treated with 1 µM rapamycin  for 1 hr to induce targeting of ALPS-ACC1-GFP-FKBP to the mitochondria outer membrane. When indicated, cells were also treated with nocodazole (2.5 µg/ml) to scatter the Golgi into mini stacks. This treatment was done 1 hr before and during rapamycin treatment. Thereafter, the cells were perforated and incubated with liposomes (250 µM lipids) as described above.The ALPS of GMAP-210 (aa 1–38) peptide and the condensed ALPS peptide were synthesized by ProteoGenix, France:WLGGLGSGLGQSLGQVGGSLASLTGQISNFTKDML (38 aa)ALPS GMAP-210: CSSWLGGLLGSLVGSLLTIFKDML (24 aa)CondALPS: CSSNote that the first methionine was mutated into a cysteine and that the sequences contain one tryptophan residue, thereby allowing determination of peptide concentration by absorbance at 280 nm and liposome partitioning by fluorescence measurement.Due to its high hydrophobicity, condALPS was only soluble in urea. Stock solutions of the two peptides (50 µM) were thus prepared in 4 M urea supplemented with 2 mM DTT.2 2.5 mM, pH 7,2 for the experiments with perforated cells. After five cycles of freezing and thawing, the liposomes were extruded sequentially through polycarbonate filters with pore diameters of 200, 100, 50 and 30 nm. Alternatively, small liposomes were obtained by sonication with a titanium tip sonicator (2 × 10 min in ice) and centrifuged for 20 min at 45,000 rpm to remove titanium debris. The liposome hydrodynamic radius (Rh) was measured by dynamic light scattering in a Dyna Pro instrument. Liposomes were stored at room temperature and used within one day.All lipids were purchased from Avanti Polar Lipids . Lipids in chloroform were mixed in a pear-shape vial and dried in a rotary evaporator. The lipid film was hydrated at 2 to 10 mM lipids either in HK buffer  for biochemical experiments or in Hepes 25 mM, KCl 115 mM, MgClNA = 1.42). The mean fluorescence intensity per pixel increased linearly with liposome concentration (from 0 to 100 µM lipids) giving a conversion factor, k:To convert the red fluorescence signal at the Golgi into a number of rhodamine-labeled liposomes, we imaged liposome suspensions in a Malassez cell (thickness 200 µm) using the same settings as that used for cell imaging  is ≈ s is the surface of one lipid (s ≈ 0.7 nm2).The number of lipids in a small liposome  and a blank spectrum was recorded. Thereafter, 0.125 µM NBD-labeled GMAP (1–375 fragment with the M1C mutation for NBD labeling  The reviewers support presentation in eLife after the following issues have been adequately addressed.1) The title of the work is: "A filter at the entrance of the Golgi that selects vesicles according to size and bulk lipid composition." The overall significance of the story would be greatly enhanced if the authors were to demonstrate the physiological significance of their findings in ALPS motif rescue experiments. This is of particular importance given reports suggesting that loss of GMAP210 can cause changes in cilia. The authors should use CRISPR to remove GMAP210 and then rescue with a version of GMAP210 that lacks the ALPS motif. This would allow them to determine if ER to Golgi traffic and intra-Golgi traffic are really dependent on the ALPS motif, and also to confirm the conclusion that the ALPS motif in GMAP-210 is the primary component responsible for liposome capture in permeabilized cells.The group of Martin Lowe has addressed this point in two papers that we cite several times in the text:http://doi.org/10.1242/jcs.166710Roboti, P., Sato, K., & Lowe, M. (2015). The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway. Journal of Cell Science, 128(8), 1595–1606. http://doi.org/10.1091/mbc.E14-10-1450Sato, K., Roboti, P., Mironov, A. A., & Lowe, M. (2015). Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210. Molecular Biology of the Cell, 26(3), 537–553. In the first paper, the authors use siRNA against GMAP-210 to assess the involvement of GMAP-210 in several traffic steps between the ER and the Golgi (ER > ERGIC > Golgi and Golgi > ER). Rescue experiments are presented in the second paper, which were conducted with various forms of GMAP-210, including ∆ALSP-GMAP-210.These studies show that the ALPS motif is necessary for the vesicle tethering activity of GMAP-210 . However, Lowe noticed: “We show here that GMAP-210–mediated tethering in cells is mediated exclusively by the N-terminal ALPS motif. On the face of it, this may appear surprising. Tethering is a specific process, with different golgins able to tether different types of vesicles , yet the ALPS motif binds to membrane lipids”. Our study addresses this issue by showing the importance of curvature and lipid unsaturation for selective vesicle capture.Of note, the efficiency of GMAP-210 silencing with siRNA is very high . Therefore, a CRISPR strategy is not mandatory at this point although this approach will be interesting to better address the physiological function of GMAP-210 in the future .As for the physiological function of GMAP-210, two KO mouse have been presented:http://doi.org/10.1371/journal.pgen.1000315Follit, J. A., Agustin, J. T. S., Xu, F., Jonassen, J. A., Samtani, R., Lo, C. W., & Pazour, G. J. (2008). The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex. PLoS Genetics, 4(12), e1000315. http://doi.org/10.1371/journal.pgen.1000315Follit, J. A., Agustin, J. T. S., Xu, F., Jonassen, J. A., Samtani, R., Lo, C. W., & Pazour, G. J. (2008). The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex. PLoS Genetics, 4(12), e1000315. In both cases, GMAP-210 deletion leads to embryonic death. Follit observed defects in cilium morphology and function through a decrease in Hedgehog signaling. Smits observed defects in skeletal tissue through decrease in perlican secretion. Although our aim was not to resolve these conflicting studies, our study suggests some hypotheses. First, the defect in perlecan secretion and glycosyslation contrasts with the normal fate of type II collagen and aggregan, which are much bigger matrix proteins. Given the ability of GMAP-210 to select vesicles based on their size, GMAP-210 deficiency should affect only matrix proteins that are incorporated into small classical transport vesicles. Second, the primary cilium has a different lipid composition than the plasma membrane in which it is embedded. Thanks to its ability to select transport vesicles according to their bulk lipid composition, GMAP-210 could help making this atypical membrane structure. These two hypotheses are presented in the new final paragraph of the Discussion.2) An unexpected observation is that the ALPS motif alone is targeted to the early Golgi, as if vesicles are accumulating there for interaction. Is this true in GMAP-210 depleted cells? Is this also true for the ALPs motif from ArfGAP1? Please elaborate. Please provide evidence that this really is the Golgi vs. a peri-Golgi compartment.cis Golgi targeting of the ALPS motif of GMAP-210”, fourth paragraph).Thank you for this suggestion. To address these points, we have conducted new experiments in which the cells were first siRNA treated for 72 hrs to deplete GMAP-210 and then transiently transfected with probes expressing the ALPS motif. Alternatively, we used cells stably expressing ArfGAP1  and again treated the cell with GMAP-210 si RNA. These experiments are presented in a new figure . In bothcis Golgi targeting of the ALPS motif of GMAP-210”, last paragraph.Since this observation does not allow us to connect the two central observations of our paper (the role of GMAP-210 in liposome capture and the targeting of the ALPS motif to the early Golgi), we performed a different experiment, which is shown in a new Figure . We perf3) The mitochondrial tethering experiment data in We acknowledge that the previous main figure lacked proper controls and that the use of only two colors  made the analysis ambiguous without further staining of membrane compartments. In addition, the supplementary figure was a jumble of different controls.As suggested by the reviewers, we now focus on two conditions in the main figure : + nocodAs for the quality of the images, we hope that the new ones are more satisfactory. However, and as explained in the legend of As for 4) Results: "GMAP-210 is responsible (for a large portion) of the ability of the Golgi" (please modify). Are there other obvious candidate proteins that can explain the residual fluorescence detected over the Golgi in GMAP-210 depleted cells? Please discuss.We have modified the sentence to temper our conclusion: “Altogether, the experiments shown in Thus, we performed a new bioinformatics search for ALPS-like motifs considering all golgins described by Munro in a recent review. The following list shows the corresponding Uniprot entry numbers:O60763, P82094, Q7Z5G4, Q8IWJ2, Q8TBA6, Q96CN9, Q08378, Q08379, Q13439, Q13948, Q14789, Q15643, Q92805As before , we found an ALPS motif in the golgin giantin (entry: Q14789|GOGB1_HUMAN Golgin subfamily). The corresponding sequence is: (2726)NKGLTAQIQSFGRSMSSLQNS(2746). This motif is localized within the coiled-coil region of this giant protein, about 500 a.a., apart from the transmembrane domain. As such, it might act as a binding site for small vesicles deeper in the golgin matrix as compared to the ALPS motif of GMAP-210, which resides at the N-terminus. However, the ALPS motif of giantin shows a moderate but significant probability to be folded as a coiled-coil. The potential role of this motif is now discussed .5) See response to point 3.6) The reader eye focuses on the organelle displaying the highest fluorescence level. For quantification, however, we did only not consider the absolute fluorescence at the Golgi but instead the Golgi/cytosol ratio. For this, we divided the fluorescence in a ‘Golgi box’ by the fluorescence in a ‘cytosol box’ of the same surface. This method gives a fair estimate of the Golgi/cytosol partitioning of the various constructs and allows detecting modest changes between constructs that do decorate the Golgi but show different levels in the cytosol. However, we acknowledge that the image contrast was not optimal to appreciate some differences in the protein level in the cytosol. We have added a supplementary figure in gray levels to better illustrate this point .


Lactose malabsorption was diagnosed using a 13C-lactose breath test. Colonic fermentation was characterized in fecal samples at baseline and after incubation with lactose for 3 h, 6 h and 24 h through a metabolomics approach using gas chromatography-mass spectrometry (GC-MS). Fecal water cytotoxicity was analyzed using a colorimetric assay. Fecal water cytotoxicity was not different between the three groups . Cluster analysis of the metabolite patterns revealed separate clusters for NLD, LM-T and LM-IT samples at baseline and after 24 h incubation with lactose. Levels of 5-methyl-2-furancarboxaldehyde were significantly higher in LM-IT and LM-T compared to NLD whereas those of an unidentified aldehyde were significantly higher in LM-IT compared to LM-T and NLD. Incubation with lactose increased short chain fatty acid (SCFA) concentrations more in LM-IT and LM-T compared to NLD. In conclusion, fermentation patterns were clearly different in NLD, LM-IT and LM-T, but not related to differences in fecal water cytotoxicity.Whether or not abdominal symptoms occur in subjects with small intestinal lactose malabsorption might depend on differences in colonic fermentation. To evaluate this hypothesis, we collected fecal samples from subjects with lactose malabsorption with abdominal complaints (LM-IT,  In about 75% of the world population the activity of this enzyme decreases after weaning (primary hypolactasia or lactase-nonpersistence), resulting in incomplete digestion of lactose and lactose malabsorption in adulthood [Humans are generally born with the ability to digest lactose, a disaccharide consisting of β-dulthood . Lactosedulthood . Very radulthood . Whereasdulthood . At preset al. (2003), lactose intolerant subjects were divided into two groups, according to the severity of their symptoms [2), carbon dioxide (CO2) and methane (CH4) [d- and l-lactate, acetate, propionate and butyrate, indicating a difference in the colonic fermentation with healthy subjects [et al. introduced the bacterial metabolic toxin hypothesis, stating that also other bacterial metabolites, such as alcohols, aldehydes, acids and ketones, resulting from carbohydrate fermentation may play a role in the pathogenesis of lactose-intolerance. These metabolites might inhibit bacterial growth and affect eukaryotic cells [In a study by Vonk symptoms . One grosymptoms . It has ne (CH4) . When insubjects . These fsubjects ,9,10. Hoic cells . In our ic cells .We hypothesize that the fermentation pattern is different in healthy controls compared to subjects with lactose-malabsorption without symptoms (lactose tolerant subjects) and subjects with symptoms (lactose-intolerant subjects). Production of different metabolites might explain toxicity and the generation of symptoms.13C-lactose breath test  and a group with no complaints  during the 13C-lactose breath test. All participants had a regular dietary pattern, were older than 18 years and had a body mass index (BMI) between 18.5 and 27 kg/m2. None of the participants were vegetarians or on a specific diet, underwent abdominal surgery (except for appendectomy) prior to the study nor had a history of chronic gastro-intestinal conditions such as inflammatory bowel disease, irritable bowel syndrome or celiac disease. None of the participants ingested anti-, pre- or probiotics one month prior to the study. Finally, all participants were free of medication affecting the gut function two weeks prior to the study.Fecal samples were collected from healthy subjects with normal lactose digestion (NLD) and subjects with low lactose digestion and lactose malabsorption (LM). The group of subjects with LM was divided into a group that presented with abdominal complaints during the 13C-lactose breath test (performed between January 2013 and December 2014), a clear description of the presence or absence of complaints experienced during the breath test to allow classification as LM-IT or LM-T and living in the proximity of Leuven to assure a rapid transport of the collected samples to the laboratory. Selected patients were sent a letter to inform them about the study and to ask them to participate. Patients that agreed to participate filled in a questionnaire on their demographic characteristics, medical history, dietary habits and drug use to check inclusion and exclusion criteria. Subjects with presumed normal lactose digestion were recruited amongst colleagues at the KU Leuven. They performed a 13C-lactose breath test to confirm normal lactose digestion and also completed the questionnaire. Eligible subjects  collected a fecal sample under anaerobic conditions.Patients with LM were selected from a database from the University Hospitals Leuven. Selection criteria were a positive This study was conducted according to the guidelines laid down in the Declaration of Helsinki and was approved by the Ethics Committee of the University Hospitals Leuven. The trial was registered at ClinicalTrial.gov . All subjects gave their written informed consent before participation.g) to prepare fecal water that was stored at −20 °C until further analysis. Another aliquot was used immediately after arrival at the laboratory to prepare 2.5% m/V fecal slurries in oxygen-free phosphate-buffered saline. One fraction of the slurry was analyzed immediately to evaluate the baseline fecal metabolite pattern. The remaining fractions were incubated for 3 h, 6 h and 24 h at 37 °C with 6.25 mg lactose or without lactose (control).Fecal samples were collected by the participants in dedicated recipients containing an Anaerocult strip  to create anaerobic conditions during transport. Within 24 h after collection of the sample, an aliquot of the fecal sample was ultracentrifuged , and ingested 50 g of 13C-labelled lactose , dissolved in 250 mL of water. Afterwards they rinsed the glass with an additional 250 mL of water. Consequently, breath samples were collected in duplicate with 30-min intervals up to 4 h after ingestion of the 13C-lactose. Subjects were also asked to report symptoms or discomfort observed during the test day.After an overnight fast, subjects collected two baseline breath samples in Exetainers2-levels were measured in one set of samples using a gas chromatograph  coupled to a thermal conductivity detector (TCD). The GC was equipped with a packed precolumn  followed by a packed column  and used nitrogen 5.0 as a carrier gas with a constant pressure of 96 kPa. Results were expressed in ppm. An increase in H2-excretion with 20 ppm above baseline was considered as an indication of bacterial metabolism of lactose. In the second set of samples, 13CO2-levels were measured using isotope-ratio mass spectrometry (IRMS)  and converted to percentage of administered dose. Normal lactose digestion was defined as a cumulative 13CO2-excretion after 4 h exceeding 14.5% of administered dose and the absence of increased H2-excretion [Hversus 2-ethylbutyrate as internal standard were calculated for all compounds. A number of metabolites, selected as markers for saccharolytic fermentation  and proteolytic fermentation  were also absolutely quantified. SCFA were determined as the sum of acetate, propionate and butyrate and branched chain fatty acids (BCFA) as the sum of isobutyrate and isovalerate. SCFA and BCFA were quantified using 2-ethylbutyrate as internal standard, whereas p-cresol was quantified versus 2,6-dimethylphenol and dimethylsulfide versus diethylsulfide.Volatile organic compounds were analyzed using a GC-MS  coupled online to a purge and trap sample preparation system as previously described . Relativ50).Cytotoxicity of fecal water was measured on HT-29 cells using a colorimetric cell viability assay based on the cleavage of a tetrazolium salt in the mitochondria of living cells to produce a colored formazan derivative, as previously described . Brieflyp < 0.05.Supervised cluster analysis ) was applied to evaluate the impact of lactose fermentation as a function of time on the metabolite patterns in the samples from NLD, LM-IT and LM-T subjects using Unscrambler software . In addition, associations between fermentation metabolites and cytotoxicity were studied. Differences in relative concentrations of volatile organic compounds and fecal water cytotoxicity between NLD, LM-IT and LM-T were evaluated using a Kruskall-Wallis-test followed by a Mann Whitney U-test. False discovery rate correction was applied to correct for multiple testing. Differences in absolute concentration of selected volatile organic compounds were evaluated using a general linear model with three between-subject factors , Group  and Treatment (with and without lactose added)). The statistical level of significance was set to 13C-lactose-breath tests have been performed in UZ Leuven. After screening, 92 subjects with a positive lactose breath test that lived in the proximity of Leuven were selected and were contacted. Sixty of them had reported symptoms whereas 32 subjects had no complaints during the test day. Finally, 11 LM-IT  and 8 LM-T  agreed to participate in the study and collected a fecal sample. In addition, 15 NLD  collected a fecal sample.Between January 2013 and December 2014, 4742 13CO2-excretion amounted to 20.5% ± 2.5% in NLD, while this was only 10.1% ± 3.3% in LM-IT and 12.7% ± 1.8% in LM-T. H2-excretion increased with 6.3 ± 7.8 ppm in NLD, 35.8 ± 47.4 ppm in LM-IT and 23.2 ± 26.8 ppm in LM-T. Two subjects in the NLD-group reported mild symptoms. No complaints were registered in the LM-T group whereas subjects in this LM-IT group reported at least one of the following symptoms: diarrhea (five subjects), flatulence (four subjects), cramps (two subjects), nausea (two subjects) and abdominal disturbances (two subjects).Cumulative p = 0.164) ; LM-IT: 15.5 (11.4–21.4); LM-T: 11.4 (6.3–12.9); Kruskall-Wallis = 0.164) .p = 0.009), isobutyrate (p = 0.028) and total BCFA (p = 0.041), were significantly higher in subjects with NLD compared to LM-IT (p = 0.038), p-cresol (p = 0.012) and total BCFA (p = 0.037) were higher in NLD subjects compared to LM-T and p-cresol was higher in LM-IT compared to LM-T. Levels of SCFA, which result from carbohydrate fermentation, were not different between the three groups.At baseline, the levels of the protein fermentation metabolites, dimethylsulfide (to LM-IT . Isobutyp = 0.00146) and LM-T (0.00173 (0.00065–0.00328); p = 0.02267), whereas the relative concentration of an unidentified aldehyde was significantly higher in LM-IT (0.0039 (0–0.00677)) compared to NLD (0 (0–0); p = 0.00468)) and LM-T (0 (0–0); p < 0.001).PLS-DA of the baseline metabolite patterns measured in the samples derived from NLD, LM-IT and LM-T revealed separate clusters for each group a. SCFA, p < 0.001; propionate: p < 0.001; butyrate: p < 0.001) and were significantly higher after incubation with lactose compared to incubation without lactose  (p < 0.001).Concentrations of SCFA increased over time during incubation in each group (acetate: = 0.003) . After iIncubation of fecal slurries with or without lactose clearly affected the metabolite patterns in the three groups . In eachp = 0.00146) and LM-T (0.00272 (0.00235–0.00379); p = 0.02267), while the relative concentration of the unidentified aldehyde was significantly higher in LM-IT (0.0073 (0–0.01161)) compared to NLD (0 (0–0); p = 0.00042)) and LM-T (0 (0–0); p < 0.001).After 24 h incubation with lactose, the metabolite patterns in the three groups were clearly separated on cluster analysis . The proIn addition, production of aldehydes and alcohols increased during fermentation of lactose . Most pr50 < 10), medium (10 < FD50 < 20) or high (FD50 > 20)). In To evaluate whether specific metabolites were associated with samples with high or low cytotoxicity, the samples were clustered according to cytotoxicity. in vitro incubation with lactose. Fecal samples from subjects with normal lactose digestion were added as a control.Not all subjects with lactose malabsorption experience undesirable effects such as bloating, cramps or abdominal pain and are to be considered as lactose-intolerant. Therefore lactose malabsorption and lactose intolerance are not synonymous and should not be interchanged. A systematic review that included the results of 18 studies found that 33%–97% of the subjects with confirmed lactose malabsorption in a hydrogen breath test, reported symptoms . Potentiet al. observed a significantly lower milk intake in intolerant lactose maldigesters and a non-significantly lower intake of cheese compared to lactose digesters and tolerant non-digesters. However, the relative protein intake was similar in the three groups [Analysis of the baseline samples revealed clear differences between the three groups of samples. Concentrations of protein fermentation metabolites were higher in subjects with normal lactose digestion compared to those with low lactose digestion. A lower degree of protein fermentation can be due to either a lower intake of protein ,19 or toe groups .Subjects that are tolerant to lactose malabsorption probably keep including dairy products in their diet. As the lactose is at least not completely digested, it enters the colon where it acts as a prebiotic and increases the level of carbohydrate fermentation. An increase in carbohydrate fermentation is a well-known mechanism of prebiotic administration to reduce the degree of protein fermentation. Indeed, the intestinal microbiota preferentially ferment carbohydrates as this process yields more energy than protein fermentation. Unfortunately, dietary intake was not assessed in this study, preventing us from confirming this hypothesis.After incubation with lactose, the production of SCFA was higher in fecal samples from subjects with lactose malabsorption (both tolerant and intolerant) compared to subjects with a normal lactose digestion, confirming previous results . AlthougIn particular, levels of alcohols and aldehydes increased clearly more in the samples with lactose than in the samples without lactose. Whereas alcohol levels generally increased as a function of time, levels of aldehydes were highest after 6 h and decreased after 24 h suggesting that aldehydes were not end products of fermentation and were further converted. Most obvious reactions are a reduction to alcohols  or an oxIn vitro studies showed that methyl furfural is mutagenic and induces strand breaks in DNA [versus non-tolerant subjects, this compound is likely not involved in the generation of symptoms in lactose-intolerant subjects. Similarly, although the levels of the unidentified aldehyde were higher in the samples from lactose maldigesters, we assume that this compound is not involved in toxicity as it is more prevalent in the samples with low to medium cytotoxicity.The levels of 5-methyl-2-furancarboxaldehyde  were significantly higher in the samples from both groups with lactose-malabsorption compared to lactose digesters. This compound occurs in pepper and other plant sources and has previously been identified in fecal samples . In addis in DNA . In the et al. introduced the bacterial metabolic toxin hypothesis, stating that bacterial metabolites resulting from carbohydrate fermentation, such as alcohols, aldehydes, acids and ketones, play a role in the pathogenesis of lactose-intolerance as these metabolites might inhibit bacterial growth and affect eukaryotic cells [Our results suggest that fecal water cytotoxicity is not the major trigger and does not explain the occurrence of abdominal complaints in intolerant lactose malabsorbers as the fecal water cytotoxicity was not different between the three groups. Campbell ic cells . A recenic cells , we founic cells ,28 confiIt is important to realize that the toxicity of fecal water was measured on the baseline fecal samples. Assessment of cytotoxicity after incubation with lactose might have been more informative but was not possible due to analytical constraints. Fecal slurries had to be used in a 1/40 dilution in order not to overload the GC column. These suspensions were too diluted for cytotoxicity experiments as the median fold dilution that induced 50% of cell death ranged from 11.4 for the samples from LM-T subjects to 16.4 for the samples from NLD subjects. Alternatively, lactose-malabsorbing subjects could have been asked to consume a lactose-rich diet during a few days prior to the sample collection. However, the amount of lactose reaching the colon may vary between subjects and hampers the interpretation of the changes in fecal metabolite profiles.in vivo. Cecal microbiota contains 100 less anaerobes than the fecal microbiota whereas facultative anaerobes constitute 25% of the microbiota in the cecum and only 1% in the feces [In addition, the fermentation pattern of lactose was evaluated using fecal microbiota which does not necessarily reflect the microbiota in the proximal part of the colon where lactose is fermented he feces . As a coin situ hybridization (FISH) and bacterial counting after plating, respectively) were not sensitive enough to detect subtle differences in microbiota composition and that current state-of-the-art techniques like next generation sequencing might be more informative.In the present study, we did not analyze the microbiota composition of the fecal samples as we hypothesized that differences in microbiota composition might explain differences in metabolite production but would not directly explain complaints. In addition, previous studies have already indicated that the microbiota composition did not differ between tolerant and intolerant subjects with lactose malabsorption  nor betwAt baseline, colonic fermentation differs in subjects with normal lactose digestion compared to subjects with lactose malabsorption with or without lactose intolerance. In addition, the functional capacity of the microbiota to process lactose is different between subjects with normal lactose digestion, lactose tolerant malabsorbers and lactose intolerant malabsorbers. However, these differences did not result in differences in fecal water cytotoxicity between the three groups. Consequently, it seems unlikely that induction of fecal water cytotoxicity is a major trigger for the occurrence of abdominal symptoms related to lactose malabsorption. Whether specific metabolites are able to trigger lactose intolerance through other mechanisms than induction of cytotoxicity remains to be investigated.